The objectives of this project are a) to develop gamma globin gene cassettes for viral vectors which could direct levels of Hb F production sufficient to inhibit in vivo sickling. b) To characterize elements which display the properties of insulators and to test whether these elements can insulate the therapeutic genes contained in viral vectors, from position effects. c) To develop new dominant selectable markers which will allow in vivo selection of transduced pluripotent stem cells. To accomplish these objectives l) we will produce a cassette containing the gamma globin genes and elements of the Locus Control Region (LCR). This LCRgamma cassette will direct therapeutic levels of gamma globin chain in the red cells; it will be of a size that can be packaged into viral vectors which also contain a gene providing selection; and it will direct- production of a gamma globin chain which can be detected in the patient's red cells using fluorescent antibodies. 2) We will use the LCRgamma globin gene construct to produce gamma globin retroviral or AAV vectors. These vectors will be used to transduce human CD34 positive progenitors and murine pluripotent stem cells. 3) We will develop approaches by which the globin genes or other therapeutic genes contained in viral vectors will be insulated from position effects at the sites of integration. This goal will be accomplished with studies of Hypersensitive Site 5 of the LCR which we have determined to contain insulator element(s). We will identify the core element(s) of the HS5 insulator; examine the function of the insulator in the context of retroviral or AAV vectors; determine the function of the element in primary cells; determine whether the insulator protects adjacent genes from being activated by the LCR. 4) In collaboration with Pilot Projects 6, 7 and 8 we will develop new dominant selectable markers conferring stem cell resistance to hydroxyurea or 5FU or busulfan. These genes will be used for production of bicistronic vectors containing the dominant selectable marker and a globin gene and they will be used for in vivo selection of transduced stem cells. The development of the recombinant genes proposed in this project will greatly increase our ability to achieve gene therapy of sickle cell disease.